【摘要】：鴨疫里默氏菌(RA)可感染鴨、鵝、火雞等多種禽類,引起一種急性敗血性或慢性傳染病,常給養(yǎng)鴨業(yè)帶來巨大的經(jīng)濟損失。為了研究RA的DnaK是否具有分子伴侶的基本特征,以及在熱脅迫等應(yīng)激條件下是否發(fā)揮作用,本文進(jìn)行了一系列的研究,獲得如下結(jié)果。1、將本實驗室分離保存的RA野毒株RA-CH-1株dnaK、dnaJ和grpE全基因序列分別克隆到表達(dá)載體pET32a(+)載體中,構(gòu)建原核表達(dá)質(zhì)粒pET32a (+)dnaK、 pET32a(+)dnaJ和pET32a (+)grpE并將其分別轉(zhuǎn)入大腸桿菌BL21中,成功表達(dá)出DnaK、 DnaJ和GrpE重組蛋白,用親和層析的方法獲得純度較高的重組蛋白。2、用超微量ATP測試盒分別測定純化的重組蛋白DnaK、DnaJ和GrpE在K+/Na+或Mg2+/Ca2+環(huán)境中水解ATP的活性。結(jié)果顯示,重組蛋白DnaJ和GrpE不具有ATPase活性；而重組蛋白DnaK具有微弱的ATPase活性且DnaJ和GrpE可增強DnaK的ATPase活性,反應(yīng)溫度50℃時DnaK的ATPase活性最高。3、用純化的DnaK蛋白免疫小鼠制備鼠抗DnaK多克隆抗體,間接ELISA檢測血清效價達(dá)到1：102400。Western blot檢測鼠抗DnaK與RA-CH-1全菌蛋白及純化的DnaK蛋白的反應(yīng)性,結(jié)果顯示該抗體能特異性地識別RA的DnaK蛋白。4、以不同應(yīng)激條件處理RA-CH-1,提取RNA，，熒光定量PCR檢測其dnaK的mRNA水平,結(jié)果表明,當(dāng)RA受到熱、H202、氨基糖苷類抗生素脅迫及滲透壓的作用時,其dnaK的轉(zhuǎn)錄水平顯著升高；受到酸堿脅迫時,dnaK的mRNA水平無明顯變化。5、根據(jù)RA的DnaK與大腸桿菌的DnaK氨基酸序列一致性達(dá)60%以上,利用λred重組的方法獲得缺失了dnaK的大腸桿菌XL1-Blue △dnaK株,同時構(gòu)建的RA DnaK原核表達(dá)載體pBAD24dnaK轉(zhuǎn)化XL1-Blue AdnaK獲得缺失回補株,在不同溫度的應(yīng)激條件下測定親本株E. coliXL1-Blue.缺失株E. coli XL1-Blue △dnaK和缺失回補株E.coliXLl-Blue AdnaK pBAD24dnaK的生長曲線,結(jié)果親本株E. coliXLl-Blue生長良好,缺失株E.coli XL1-Blue AdnaK在42℃時不生長,而回補了RA DnaK的E. coliXL1-Blue AdnaK pBAD24dnaK生長速率得到一定的恢復(fù)。表明RADnaK不僅能在一定程度上增強大腸桿菌XL1-Blue△dnaK對溫度的耐受,在RA受到各種理化因子的刺激時,可能也扮演非常重要的角色。綜上,RA的DnaK蛋白具有ATPase活性,DnaJ和GrpE能有效提高其ATPase的活性,且DnaK在RA抗高溫、抗氧化、抗?jié)B透壓和抗氨基糖苷類抗生素等應(yīng)激時具有重要的作用,但在抗酸堿協(xié)迫時并不發(fā)揮功能；RA DnaK在一定程度上能替代大腸桿菌的DnaK在熱休克等應(yīng)激反應(yīng)中增強其耐受性。
[Abstract]:Riemerella (RA) can infect ducks, geese, turkeys and other birds, causing an acute septicemia or chronic infectious disease, which often brings great economic losses to the duck industry. In order to study whether the DnaK of RA has the basic characteristics of molecular chaperone and whether it functions under stress conditions such as heat stress, a series of studies have been carried out in this paper, and the following results have been obtained. The dnaK,dnaJ and grpE gene sequences of RA field virus strain RA-CH-1 isolated in our laboratory were cloned into the expression vector pET32a () respectively, and the prokaryotic expression plasmid pET32a () dnaK, was constructed. PET32a () dnaJ and pET32a () grpE were transformed into E. coli BL21, respectively. The recombinant proteins of DnaK, DnaJ and GrpE were successfully expressed, and the recombinant proteins with higher purity were obtained by affinity chromatography. The activity of purified recombinant proteins DnaK,DnaJ and GrpE to hydrolyze ATP in K / Na or Mg2 / Ca2 environment was determined by ultra-micro ATP test box. The results showed that the recombinant proteins DnaJ and GrpE had no ATPase activity. The recombinant protein DnaK had weak ATPase activity and DnaJ and GrpE could enhance the ATPase activity of DnaK. The ATPase activity of DnaK was the highest at reaction temperature of 50 鈩

本文編號：2472304